Comparison of target labeling methods for use with Affymetrix GeneChips

The only notable difference between kits was in the yield of cRNA target synthesized during in vitro transcription, where the BioArray assay had to be repeated several times in order to have sufficient target. However, each kit resulted in comparable signal and detection calls when hybridized to the Affymetrix GeneChip.These 3 one-cycle labeling kits produce comparable hybridization results. This provides users with several kit options and flexibility when using the Affymetrix system.There are several commercially available one-cycle labeling kits that generate targets for use with Affymetrix GeneChip technology. Commercial labeling kits are valuable because they eliminate the need for individual laboratories to optimize methods, saving both time and resources. They also allow better cross-comparison of results generated from different laboratories. However, this assumes that all approaches produce comparable labeled targets. Given the widespread use of several labeling kits with the Affymetrix technology, we evaluated cRNA target synthesis, labeling and hybridization results using 3 different one-cycle linear amplification labeling kits. These were the One-Cycle Target Labeling Assay from Affymetrix (One-Cycle), the BioArray？ RNA Amplification and Labeling System from Enzo Life Sciences (BioArray), and the Superscript？ RNA Amplification System from Invitrogen Life Technologies (Superscript). Several steps for synthesizing labeled target are identical for each kit. For example, each kit uses reverse transcriptase with an anchored oligo(dT) primer containing a T7 promoter to synthesize first-strand cDNA. Then, following second strand synthesis, the cDNA templates are amplified via the Eberwine isothermal protocol [1]. Some of the differences include the biotinylated nucleotides in the in vitro transcription (IVT) reaction. The BioArray kit uses two biotinylated nucleotides; One-Cycle, a biotinylated pseudo-nucleotide and the Superscript kit a single biotinylated nucl